JPH0689037B2 - GnRH antagonist - Google Patents

Abstract

Peptides which inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The peptides have the structure: X- beta -D-2NAL-R2-D-Trp-Ser-Arg-R6-R7-Arg-Pro-R10 wherein X is hydrogen or Ac; R2 is Cl-D-Phe, F-D-Phe or C alpha Me-Cl-D-Phe; R6 is D-Tyr, beta -D-2NAL or D-Arg; R7 is Leu or N alpha Me-Leu; and R10 is Gly-NH2, NHCH2CH3 or D-Ala-NH2.

Description

The present invention relates to peptides that suppress the release of gonadotropic hormones from the pituitary gland of mammals, including humans, and a medicament for regulating ovulation and / or steroid secretion from the gonads. It relates to a composition. More particularly, the invention relates to peptides that inhibit gonad function and the release of steroidal hormones (progesterone and testosterone).

BACKGROUND OF THE INVENTION One of the hormones produced in the hypothalamus acts as a factor in inducing the secretion of gonadotrophic hormones (especially LH), which will be referred to herein as GnRH, which also Also called LH-RH and LRF. GnR
H has been isolated and has the following structure: p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-G
It was characterized as a decapeptide with ly-NH ₂ .

A peptide is a compound containing two or more amino acids, in which the carboxyl group of one amino acid is bonded to the amino group of the other amino acid. The formula for GnRH shown above follows the conventional convention for peptides, with the amino group on the left and the carboxyl group on the right. Amino acid residue positions are identified by numbering amino acid residues from left to right. In the case of GnRH, the hydroxyl part of the carboxyl group of glycine is replaced with an amino group (NH ₂ ). Abbreviations for individual amino acid residues are conventional and are based on the common name of amino acids, such as p-Gl.
u is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Arg is arginine, Pro is proline, Phe is phenylalanine, and Ala is alanine. These amino acids occur naturally with valine, isoleucine, threonine, lysine, aspartic acid, asparagine, glutamine, cysteine, methionine and proline. That is, it is a normal amino acid that constitutes a protein. Except for glycine, the amino acids of the peptides of the invention are in the L-configuration unless otherwise indicated.

There are many reasons for wanting to suppress ovulation in female mammals,
GnRH antagonizes the normal function of GnRH to suppress ovulation
Analogs have been administered. For this reason GnRH analogs that antagonize GnRH are being investigated for their potential use as contraceptives or for controlling the time of pregnancy. Antagonists of this kind have also been found to be useful in regulating the secretion of gonadal hormones in male mammals and can be used as male contraceptives.
It is desired to provide peptides that strongly antagonize GnRH produced in vivo and suppress the secretion of LH and the release of steroids from the gonads of mammals.

SUMMARY OF THE INVENTION The present invention provides peptides that inhibit the release of gonadotropic hormones in mammals, including humans, and methods of inhibiting the release of steroids from the gonads of male and female mammals. The improved GnRH analogs antagonize GnRH and have an inhibitory effect on the mammalian reproductive process. These analogs can be used to inhibit the production of gonadotropins and sex hormones under various circumstances such as precocious puberty, hormone-dependent tumors, dysmenorrhea and endometriosis .

In general, according to the present invention, peptides were synthesized that potently inhibit the secretion of gonadotropic hormones from the pituitary gland of mammals, including humans, and / or inhibit the release of steroids from the gonads. . These peptides are GnRHs themselves, such as 1-position substitutions such as β- (2-naphthyl) D-alanine (hereinafter β-D-2NAL), 3-position substitutions such as D-Trp, Tyr Instead, there is a 5-position substitution of Arg or D-Arg, a D-isomer substitution of the 6-position as is generally known in the art, and a specific substituent at the 2-position. The 1-position substituent may be modified such that its α-amino group comprises acetyl (Ac) or acryl (Acr), or it may be unmodified. The modified D-Phe is present at the 2-position and, as a result of the specific modification present on the benzene ring, provides increased antagonism, especially when methylating the α carbon atom. The only substitution for a hydrogen atom is at the loose or 4-position, with chloro and fluoro being preferred. 2 for this D-Phe residue
One substitution, ie one α carbon atom and one substitution on the benzene ring (eg C ^α Me -4Cl-D-Phe) is preferred. D-Tyr or D-Arg is advantageously present in the 6-position. Substitutions at positions 7 and 10 are optional.

These peptides are very often called GnRH antagonists because they very strongly suppress the release of LH. These peptides are effective in blocking ovulation in female mammals when administered at very low levels during proestrus and in causing fertilized egg absorption when administered shortly after pregnancy. These peptides are also effective for contraceptive treatment of male mammals.

DISCLOSURE OF THE INVENTION The peptides of the present invention are specifically described by the following formula: XR ₁ -R ₂ -R ₃ -Ser-R ₅ -R ₆ -R ₇ -Arg-Pro-R ₁₀ (where X is hydrogen, Acr Or Ac; R ₁ is β-D-2
NAL, dehydroPro or 4Cl-D-Phe; R ₂ is Cl-
R-is D-Phe, F-D-Phe or C ^α Me-Cl-D-Phe; R ₃ is D-Trp or β-D-2NAL; R ₅ is Arg or D-Arg; R ₆ Is D-Tyr, β-D-2NAL or D-Arg; R ₇ is Leu or NML; and R ₁₀ is
Gly-NH _2, represented by NHCH ₂ CH ₃ or D-Ala-NH _2).

β-D-NAL means the D-isomer of alanine in which the β carbon atom is substituted with naphthyl, and is also referred to as 3-D-NAL. Preferably β-D-2NAL is used, which means that its β-carbon atom is attached to naphthalene at the 2-position of its ring structure. However, β-D-1NAL is also considered equivalent. NM
L means that there is a methyl substitution in the α-amino group of Leu.

The peptides of the present invention can be synthesized by a basic solution synthesis method or a solid phase synthesis method using a chloromethylated resin, a methylbenzhydrylamine (MBHA) resin or a benzhydrylamine (BHA) resin. The solid phase synthesis method is the method described in detail in US Pat. No. 4,216,193, and is carried out by sequentially adding amino acids to the peptide chain. Preferably side chain protecting groups well known in the art are added to Ser, Tyr and Arg and optionally also TrP, after which these amino acids are coupled to a resin bound peptide chain. . This type of method gives well protected peptide resin intermediates.

The intermediate of the present invention has the following formula: X ¹ -R ₁ -R ₂ -R ₃ (X ² ) -Ser- (X ³ ) -R ₅ (X ⁵ ) -R ₆ (X ⁴ or X ⁵ ) -R _7-
Represented by Arg (X ⁵ ) -Pro-X ⁶ , where X ¹ is an α-amino protecting group known to be useful in the art of stepwise synthesis of polypeptides and is intended When X in the peptide composition is a specific acyl group, that group can also be used as a protecting group. The α-amino protecting group included in X ¹ includes (1) an acyl-type protecting group such as formyl (For), trifluoroacetyl, phthalyl, p-toluenesulfonyl (To).
s), benzoyl (Bz), benzenesulfonyl, o-nitrophenylsulfenyl (Nps), tritylsulfenyl, o-nitrophenoxyacetyl, acrylic (Ac
r), chloroacetyl, acetyl (Ac) and γ-chlorobutyryl; (2) aromatic urethane type protecting groups such as benzyloxycarbonyl (Z) and p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromo. Benzyloxycarbonyl and p-
Substituted benzyloxycarbonyl such as methoxybenzyloxycarbonyl; (3) Aliphatic urethane type protecting groups such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cyclo Alkyl urethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl;
(5) Thiourethane type protecting groups such as phenylthiocarbonyl; (6) Alkyl type protecting groups such as allyl (Al
y), triphenylmethyl (trityl) and benzyl (Bzl); (7) trialkylsilane type protecting groups such as trimethylsilane. A preferred α-amino protecting group when X is hydrogen is Boc.

X ² is hydrogen or a protecting group for the indole nitrogen (eg formyl or benzyl). However, it is not necessary to protect Trp in many syntheses.

X ³ is hydrogen or a protecting group for the alcoholic hydroxyl group of Ser, acetyl, benzoyl, tetrahydropyranyl, t-butyl, trityl, benzyl and 2,
It is selected from the group consisting of 6-dichlorobenzyl. Benzyl is preferred.

X ⁴ is hydrogen or a phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, t-butyl, trityl, benzyl, benzyloxycarbonyl, 4-bromobenzyloxycarbonyl and 2,6-dichlorobenzyl. Is a protective group of. 2,6-dichlorobenzyl is preferred.

X ⁵ is a protecting group for the nitrogen atom of Arg, nitro, To
s, benzyloxycarbonyl, adamantyloxycarbonyl and Boc. Alternatively, X ⁵ may be hydrogen, which means that there is no protecting group on the side chain nitrogen atom of arginine. Tos
Is preferred.

X ⁶ is Gly-O-CH ₂ - [resin support]; O-CH ₂ - [resin support]; D-Ala-O-CH 2 - [resin support]; Gly-NH-
[Resin support]; D-Ala-NH- [Resin support]; and G
selected from the group consisting of OH, ester, amide and hydrazide of ly or D-Ala, or OH, ester, amide and hydrazide directly bonded to Pro.

Criteria for selecting side chain protecting groups for X ² to X ⁵ is stable to the reagent under the reaction conditions selected for removing the α- amino protecting group at each step of the protecting group is synthesized It has to be. The side chain protecting group must not be cleaved off under coupling conditions, and the protecting group must be removable under reaction conditions that do not denature the peptide chain at the completion of the synthesis of the intended amino acid sequence.

X ⁶ is Gly-O-CH ₂ - [resin support], D-Ala-O-CH 2 -
In the case of [resin support] or O—CH ₂ — [resin support], one ester moiety among many functional groups of the polystyrene resin support is indicated. X ⁶ is Gly-NH-
In the case of [resin support] or D-Ala-NH- [resin support], Gly or D-Ala is bound to BHA by an amide bond.
Bound to resin or MBHA resin.

When X is Ac or Acr in the final formula, it is added to the D-NAL by adding Ac or Acr before coupling the last amino acid D-NAL to the peptide chain.
Can be used as the X ¹ protecting group for the α-amino group. However, the reaction was carried out using a peptide supported on a resin (one in which the protecting group of the α-amino group was removed while the side chain group was protected) and dicyclohexylcarbodiimide (DC
Advantageously, it is carried out by reacting acetic acid in the presence of C), more preferably acetic anhydride, or by any other suitable reaction well known in the art.

The fully protected peptide is cleaved from the chloromethylated resin support by ammolysis to produce the fully protected amide intermediate. Removal of the protecting group of this peptide can be carried out with hydrofluoric acid (HF) at 0 ° C., similar to the cleavage of the peptide from the benzhydrylamine resin. Advantageously anisole is added to the peptide prior to treatment with HF. After removal of HF under vacuum, the peptide cleaved from the resin and freed of protecting groups is treated with ether, decanted, extracted with dilute acetic acid and lyophilized.

The peptide was purified by ion exchange chromatography on a CMC column, followed by partition chromatography on a column packed with Sephadex G-25 using the following eluents: n-butanol; 0.1N acetic acid (1: 1 volume ratio). This can be done by E.I. or by using HPLC well known in the art.

The invention thus also the following _{formula: XR 1 -R 2 -R 3 -Ser} -R 5 -R 6 -R 7 in -Arg-Pro-R ₁₀ (wherein, X is hydrogen, Acr or Ac; R ₁ Is β-D-2
NAL, dehydroPro or 4Cl-D-Phe; R ₂ is Cl-
R-is D-Phe, F-D-Phe or C ^α Me-Cl-D-Phe; R ₃ is D-Trp or β-D-2NAL; R ₅ is Arg or D-Arg; R ₆ Is D-Tyr, β-D-2NAL or D-Arg; R ₇ is Leu or NML; and R ₁₀ is
Gly-NH _2, NHCH provides a peptide or a manufacturing method of a non-toxic salt represented by ₂ CH ₃ or D-Ala-NH _2), the method comprising (a) the following formula: X _¹ -R ¹ - ^{_{R 2 -R 3 (X 2)}} -Ser- (X 3) -R 5 (X 5) -R 6 (X 4 or X ⁵⁾ -R ₇ -
Arg (X ⁵ ) -Pro-X ⁶ [wherein X ¹ is hydrogen or an α-amino protecting group; X ² is hydrogen or a protecting group for the indole nitrogen of Trp; X ³ is hydrogen or Ser. X ⁴ is a protecting group for a hydrogen or Tyr phenolic hydroxyl group; X ⁵ is a protecting group for a nitrogen atom of hydrogen or Arg; and X ⁶ is G
ly-O-CH ₂ - (resin support), O-CH ₂ - (resin support), D-Ala-O- CH 2 - ( resin support), Gly-NH-
(Resin support), D-Ala-NH- (resin support), Gly-
NH ₂ , NHCH ₂ CH ₃ , and an ester of Gly or D-Ala,
Selected from the group consisting of amides and hydrazides]. (B) Group X ¹
Removing one or more of X ⁵ and / or cleaving from the resin support contained in X ⁶ and optionally converting the resulting peptide to its non-toxic salt.

The peptides of the present invention are effective in suppressing ovulation of female rats when administered at a dose of 100 μg or less per kg body weight at about noon on the day of proestrus. For long-term ovulation suppression, it may be necessary to use dosages in the range of about 0.1-2.5 mg / kg body weight. These antagonists are also effective as contraceptives when regularly administered to male mammals. Since these compounds may reduce testosterone levels, which is an undesirable consequence for normal, sexually active males, it is possible to administer a replacement dose of testosterone with a GnRH antagonist. Would be appropriate. These antagonists can also be used in regulating the production of gonadotropins and sex steroids for other purposes as indicated above.

Examples The following formula: Ac-R ₁ -R ₂ -R ₃ -Ser-Arg-R ₆ -Leu-Arg-Pro-R ₁₀ Peptides as shown in Table 1 as shown in the solid phase described above. Manufactured by the method.

Table 1 As one example, [Ac-β-D-2NAL ¹ , C ^α Me-4Cl-
^{D-Phe 2, D-Trp} 3, Arg 5, D-Arg 6, D-Ala 10 ] -G
A typical solid phase synthesis method of the above peptide No. 1 described as nRH will be described below. This peptide has the following formula: Ac-β-D-2NAL-CMe-4Cl-D-Phe-D-Trp-Ser.
Represented by -Arg-D-Arg-Leu- Arg-Pro-D-Aia-NH 2.

CH ₂ Cl ₂ using BHA resin and Boc-protected D-Ala with 3-fold excess of Boc derivative and DCC as activating reagent.
The resin was capped at room temperature for 2 hours.
The alanine residue was attached to the BHA resin with an amide bond.

After coupling of each amino acid residue, washing, removal of protecting groups and coupling of the next amino acid residue was first carried out on an automated synthesizer using about 5 g of resin according to the following schedule.

After step 13, if the synthesis is done manually, take an aliquot for the ninhydrin test and, if the test is negative, step 1 for the coupling of the next amino acid.
, And if the test is positive or slightly positive, repeat steps 9-13 and back.

The above schedule was used to couple each amino acid of the peptides of the invention after attaching the first amino acid. N ^α Boc-β-D-2 NAL was prepared by methods well known in the art such as those described in detail in US Pat. No. 4,234,571 issued Nov. 18, 1980. The side chain of Arg was protected with Tos. OBzl was used as a side chain protecting group for the hydroxyl group of Ser, and 2,6-dichlorobenzyl was used as a side chain protecting group for the hydroxyl group of D-Tyr in synthesizing peptide No. 2. . Trp has no protection. M ^α Boc-β-D-2NAL was introduced as the last amino acid. Since both Boc-Arg (Tos) and Boc-D-Trp showed low solubility in _{CH 2 Cl 2, DMF: CH} 2 Cl 2
The mixture was used to couple.

After removing the protecting group for the α-amino group at the N-terminus,
Acetylation was carried out using a large excess of acetic anhydride in dichloromethane. Cleavage of the peptide from the resin and complete removal of the side-chain protecting groups was done very easily with HF at 0 ° C. Prior to the HF treatment, anisole was added as a skim bender. After removing HF under vacuum, the resin was extracted with 50% acetic acid, and the washing solution was freeze-dried to obtain a crude peptide powder.

Then, ion exchange chromatography purification of the CMC of the peptide (WhatmanCM32, using a gradient of mixed solvent 0.05~0.3MNH ₄ OAc methanol / water 50:50), followed by the following dissolution agent: n-butanol; 0.1 Performed by partition chromatography on a gel percolation column with N-acetic acid (1: 1 volume ratio).

The peptide was found to be homogeneous by thin layer chromatography using a variety of different solvent systems and by reversed phase high pressure liquid chromatography using aqueous trityl ammonium phosphate and acetonitrile. Amino acid analysis of the resulting purified peptide was in agreement with the prepared structural formula, substantially indicating the integer value for each amino acid of the peptide chain. The optical rotation is measured with a photoelectric polarimeter, and ▲ [α] ²² _D ▼ ＝
It was −2.2 ° ± 1 (c = 1, 50% acetic acid).

The described peptides were tested in vivo to confirm their effectiveness in inhibiting ovulation in female rats. In this study, a specific number of mature female rats of the Spragut-Dawley variety (ie 10 animals each weighing 225-250 g) were injected with 10 μg of the peptide in corn oil at about noon on the day of proestrus. Preestrus is the afternoon before estrus (ovulation). Another female rat group was used as a control, and this group received no peptide. Each control female rat ovulated during oestrus, but none of the treated rats ovulated. As a result, the peptide was considered to be significantly effective at inhibiting ovulation in female rats at very low doses, and the peptide was considered to be completely effective at doses of about 10 μg. Additional studies were conducted at lower or higher doses and the results are shown in Table II below. In the table, all optical rotation values are c =
It was measured under the condition of 1.50% acetic acid.

The following formula: Ac-D-2NAL-4Cl-D-Phe-D-3PAL-Ser-Arg-D
The peptide -Glu (AA) -Leu-Arg- Pro-D-Ala-NH 2 (AA = anisole adduction), was subjected to toxicity testing.

Using 12 rats of each sex, according to GMP standards, 3
A 0-day test was conducted. The peptide dose used was 5
It was 0, 250 or 1250 μg / kg body weight. Peptides were injected subcutaneously for 30 days. Rats were dissected on day 31 for serological, hematological and microscopic and gross histological studies.

All animals were tolerated at all doses with no apparent systemic effect. All rats survived the experimental period.

The same test was then performed on this peptide for 90 days and all animals were tolerated at all doses with no apparent systemic effect. Also, all rats survived the experimental period.

All peptides are believed to be effective in suppressing ovulation in female mammals at very low doses. These peptides are pharmaceutically acceptable non-toxic salts, such as acid addition salts or metal complexes (eg complexes with zinc, barium, calcium, magnesium or aluminum, which complexes are considered herein as addition salts), Alternatively, it can be administered in the form of a combination of the two. Examples of acid addition salts include hydrochloride, hydrobromide, sulfate, phosphate, nitrate, acid salt, fumarate, gluconate, tannate, maleate, acetate, citrate, Benzoate,
Examples include succinate, alginate, malate, ascorbate and tartrate. When the active ingredient is administered in the form of tablets, tablets may be provided with binders (eg tragacanth,
Corn starch or gelatin); disintegrants (eg alginic acid); and pharmaceutically acceptable diluents including lubricants (eg magnesium stearate) can be included. If administration in liquid form is desired, sweetening and / or flavoring agents may be used as part of the pharmaceutically acceptable diluent, and isotonic saline or phosphate buffered saline Internal administration can also be performed.

The pharmaceutical composition will normally contain the peptide together with a conventional pharmaceutically acceptable carrier. Generally, the dose will be about 1 to 100 μg of peptide per kg of patient's body weight when administered intravenously, and the oral dose may be higher. After all, the treatment of patients with these peptides is similar to other Gn
It is done in the same way as clinical treatment with RH antagonists.

These peptides are used to achieve fertility suppression and also when reversible suppression of gonadal activity is required, e.g. for the management of precocious puberty or during radiation or chemotherapy. It is administered intravenously, subcutaneously, intramuscularly, orally, intranasally or intravaginally. Effective doses will vary with the form of administration and the type of mammal being treated. An example of one representative dosage form is saline containing the peptide, which solution is about 0.1-2 kg / kg body weight.
It is administered to provide a dose in the range of 5 mg. Oral administration of peptides is given in either solid or liquid form.

While this invention has been described in terms of a preferred embodiment, changes and modifications as would be apparent to one of ordinary skill in the art may be made without departing from the scope of the invention as set forth in the claims. You should understand that you get.
For example, other substitutions well known in the art that do not significantly reduce the efficacy of the peptide may also be used for the peptides of the invention. As an example, Gly-O
_{CH 3, Gly-OCH 2 CH} 3, Gly-NHNH 2 and Sar-NH ₂ (Sar =
Sarcosine) is considered the equivalent of the amino acid residues specified for R ₁₀ .

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Jijan Edouard Frederik Libere California, United States 92037, La Jolla, Bratsk Gold Road 9674 (72) Inventor Wiley Walker Veil Giunia United States California Province 92037, La Jolla, Valdez 1643

Claims (18)

[Claims] Claims: XR ₁ -R ₂ -R ₃ -Ser-R ₅ -R ₆ -Leu-Arg-Pro-R ₁₀ (wherein X is hydrogen or Ac; R ₁ is β- D-2NAL,
DehydroPro or 4Cl-D-Phe; R ₂ is Cl-D-P
he, FD-Phe or C ^α Me-Cl-D-Phe; R ₃
Is D-Trp or β-D-2NAL; R ₅ is Arg or D
It is -Arg; R ₆ is D-Tyr, β-D- 2NAL or D-Ar
It is g; and the peptide or its non-toxic salts R ₁₀ is represented by a Gly-NH ₂ or D-Ala-NH _2). 2. The peptide according to claim 1, wherein R ₅ is Arg. 3. X is Ac and R ₁ is β-D-2NAL,
The peptide according to claim 1 or 2, wherein R ₃ is D-Trp. 4. The peptide according to any one of claims 1 to 3, wherein R ₂ is C ^α Me-4-Cl-D-Phe. 5. The peptide according to claim 4, wherein R ₆ is D-Arg. 6. The peptide according to claim 4, wherein R ₆ is D-Tyr. 7. The peptide according to any one of claims 1 to 3, wherein R ₆ is β-D-2NAL. 8. The peptide according to any one of claims 1 to 3, wherein R ₂ is 4Cl-D-Phe. 9. The peptide according to any one of claims 1 to 8, wherein R ₁₀ is D-Ala-NH ₂ . 10. R ₅ is Arg, R ₁ , R ₂ , R ₃ , R ₆ and R
The peptide according to claim 1, wherein ₁₀ is any of the following. 11. The following formula: XR ₁ -R ₂ -R ₃ -Ser-R ₅ -R ₆ -Leu-Arg-Pro-R ₁₀ (wherein X is hydrogen or Ac; R ₁ is β- D-2NAL,
DehydroPro or 4Cl-D-Phe; R ₂ is Cl-D-P
he, FD-Phe or C ^α Me-Cl-D-Phe; R ₃
Is D-Trp or β-D-2NAL; R ₅ is Arg or D
It is -Arg; R ₆ is D-Tyr, β-D- 2NAL or D-Ar
g; and R ₁₀ is Gly-NH ₂ or D-Ala-NH ₂ ) or a non-toxic salt thereof as an active ingredient in an effective amount, and further pharmaceutically acceptable non-toxic A pharmaceutical composition for regulating the secretion of gonadotropin containing a sex diluent. 12. The following formula: XR ₁ -R ₂ -R ₃ -Ser-R ₅ -R ₆ -Leu-Arg-Pro-R ₁₀ (wherein X is hydrogen or Ac; R ₁ is β- D-2NAL,
DehydroPro or 4Cl-D-Phe; R ₂ is Cl-D-P
he, FD-Phe or C ^α Me-Cl-D-Phe; R ₃
Is D-Trp or β-D-2NAL; R ₅ is Arg or D
It is -Arg; R ₆ is D-Tyr, β-D- 2NAL or D-Ar
g; and R ₁₀ is Gly-NH ₂ or D-Ala-NH ₂ ) or an ovulatory and / or gonadal steroid secretion comprising an effective amount of a non-toxic salt thereof. A pharmaceutical composition for. 13. R ₂ is C ^α Me-4-Cl-D-Phe, R 2
13. The composition according to claim 12, wherein ₃ is D-Trp. 14. The composition according to claim 12 or 13, wherein R ₁ is β-D-2NAL. 15. The composition according to claim 14, wherein R ₅ is Arg and R ₆ is D-Arg. 16. The composition according to claim 14, wherein R ₅ is Arg and R ₆ is D-Tyr. 17. The composition according to any one of claims 12 to 16, wherein R ₁₀ is D-Ala-NH ₂ . 18. The composition according to any one of claims 12 to 17, wherein X is Ac.